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Current Research and Scholarly Interests

My research over last 20 years is mainly focused on development and research of clinical protocols to assess functional status of different types of small diameter nerve fibers in animals, humans and patients with chronic pain. This research was initially focused on development a model of laser induced acute pain to prove a low energy laser pain therapy. The low energy pain therapy is still magic. However, This research lead us to development of a portable diode laser (DL) that is capable of evoking a variety of skin sensations such as: warmth, hot, touch, pricking and burning pain. I set up my private laboratory (Lasmed LLC) in 2003 and performed studies that confirmed selective activation of A delta or C nerve fibers and trpv1 or trpv2 positive cells in collaboration with Dr. David Yeomans and several other leading pain research groups. This R&D was supported by several NIH funded grants. The correlation of modality of sensation with particular nerve fiber type was verified by electrophysiological recording. DL stimulation has unique properties compared to contact and radiant heat or CO2 laser stimulation. Infrared diode laser radiation that we use for stimulation is highly controlled and the energy mainly absorbed in skin by H2O molecules. The absorption coefficient is about 0.4 cm-1 for 980 nm of DL wavelength. Such low absorption practically eliminates the differences between heating humans’ and animals’ skins; and superficially or sub-dermally cutaneous tissue is evenly activated. These properties of DL radiation are critical for translational pain research, because they allow similar DL fiber type selective (A delta vs. C fibers) protocols for preclinical and clinical studies. Through use of our DL protocols we discovered that: 1) the same type of trpv1 expressed cutaneous nociceptors are responsible for the pain in glabrous and hair skin near the pain threshold; 2) the depletion of trpv1 expressed nerve fibers leads to complete loss of acute and inflammatory pain sensitivity. Recently we tested patients with diabetic and other painful neuropathies and found that our A delta selective protocol predicted an expected increase of pain threshold, but the C-fibers selective protocol showed an unexpected result; similar laser intensity is required to stimulate C nociceptors near pain thresholds in healthy subjects and patients with diabetic painful neuropathy. We tested a few hypotheses and finally concluded that, within the framework of superficially located and highly degraded C polymodal fibers, we were not able to explain this phenomenon and deep located silent or C mechano -insensitive nerve fibers is likely involved.

Currently I am focused on application of our DLss protocols for diagnosis of diabetic and chemo induced peripheral neuropathy and study of underline peripheral mechanisms and source of these neuropathies. We expected that these studies allowed us developing predictive biomarkers of painful neuropathies and a diode laser treatment to knock out a cutaneous source of a peripheral neuropathic pain.

Abstract

TRPV1 is expressed in a subpopulation of myelinated Aδ and unmyelinated C-fibers. TRPV1+ fibers are essential for the transmission of nociceptive thermal stimuli and for the establishment and maintenance of inflammatory hyperalgesia. We have previously shown that high-power, short-duration pulses from an infrared diode laser are capable of predominantly activating cutaneous TRPV1+ Aδ-fibers. Here we show that stimulating either subtype of TRPV1+ fiber in the paw during carrageenan-induced inflammation or following hind-paw incision elicits pronounced hyperalgesic responses, including prolonged paw guarding. The ultrapotent TRPV1 agonist resiniferatoxin (RTX) dose-dependently deactivates TRPV1+ fibers and blocks thermal nociceptive responses in baseline or inflamed conditions. Injecting sufficient doses of RTX peripherally renders animals unresponsive to laser stimulation even at the point of acute thermal skin damage. In contrast, Trpv1-/- mice, which are generally unresponsive to noxious thermal stimuli at lower power settings, exhibit withdrawal responses and inflammation-induced sensitization using high-power, short duration Aδ stimuli. In rats, systemic morphine suppresses paw withdrawal, inflammatory guarding, and hyperalgesia in a dose-dependent fashion using the same Aδ stimuli. The qualitative intensity of Aδ responses, the leftward shift of the stimulus-response curve, the increased guarding behaviors during carrageenan inflammation or after incision, and the reduction of Aδ responses with morphine suggest multiple roles for TRPV1+ Aδ fibers in nociceptive processes and their modulation of pathological pain conditions.

Abstract

Sensory function of small peripheral nerve fiber was assessed by means of quantitative sensory testing (QST) during which sensory stimulation was provided using diode laser (DL) in patients suffering from painful neuropathy (PN) and compared with symptom-free healthy controls (HC). Based on previous research work using DL stimulation, parameters that demonstrated safe and specific activation of A-delta, which were distinct from stimulation parameters for the activation of C-fibers, were utilized in this study. Results of this study demonstrated that this differential activation pointed to the impaired function of A-delta fibers while C-fiber function was unaffected. Stimulation of HC reproduced previously published results, and stimulation during this study was safe also without any dermal effect in patients with PN and in HC. Parameters used in this study were demonstrated in previous preclinical rodent study identical differential effect on activation of A-delta and C-fibers, and as such, DL is an ideal tool for translational pain research where specific activation of A-delta or C-fibers, or both, is required.

Abstract

We previously demonstrated that genetic and/or pharmacological ablation of the TRPV1+/peptidergic or the MrgprD+/non-peptidergic subset of nociceptors produced selective, modality-specific deficits in the behavioural responses to heat and mechanical stimuli, respectively. To assess whether this modality-specific contribution is also manifest at the level of spinal cord neuron responsiveness, here we made extracellular recordings from lumbar dorsal horn neurons of the mouse in response to graded thermal and mechanical stimulation. We found that, following intrathecal injection of capsaicin to eliminate the central terminals of TRPV1+ nociceptors, neurons in the region of laminae I and V of the spinal cord lost responsiveness to noxious heat (whether generated by a contact heat probe or diode laser), with no change in their response to noxious mechanical stimulation. In contrast, ablation of MrgprD+ afferents did not alter the response to noxious heat, but reduced the firing of superficial dorsal horn nociceptive-specific neurons in response to graded mechanical stimulation and decreased the relative number of wide dynamic range neurons that were exclusively mechanosensitive. Neither ablation procedure reduced the number of dorsal horn neurons that responded to noxious cold. These findings support the conclusion that TRPV1+ nociceptors are necessary and probably sufficient for noxious heat activation of dorsal horn neurons and that, despite their polymodal properties, TRPV1+ and MrgprD+ nociceptors provide modality-specific contributions to the response properties of spinal cord neurons.

Abstract

Two main classes of peripheral sensory neurons contribute to thermal pain sensitivity: the unmyelinated C fibers and thinly myelinated A? fibers. These two fiber types may differentially underlie different clinical pain states and distinctions in the efficacy of analgesic treatments. Methods of differentially testing C and A? thermal pain are widely used in animal experimentation, but these methods are not optimal for human volunteer and patient use. Thus, this project aimed to provide psychophysical and electrophysiological evidence that whether different protocols of infrared diode laser stimulation, which allows for direct activation of nociceptive terminals deep in the skin, could differentially activate A? or C fiber thermonociceptors in volunteers.Short (60 ms), high intensity laser pulses (SP) evoked monomodal "pricking" pain which was not enhanced by topical capsaicin, whereas longer, lower power pulses (LP) evoked monomodal "burning" pain which was enhanced by topical capsaicin. SP also produced cortical evoked EEG potentials consistent with A? mediation, the amplitude of which was directly correlated with pain intensity but was not affected by topical capsaicin. LP also produced a distinct evoked potential pattern the amplitude of which was also correlated with pain intensity, which was enhanced by topical capsaicin, and the latency of which could be used to estimate the conduction velocity of the mediating nociceptive fibers.Psychophysical and electrophysiological data were consistent with the ability of short high intensity infrared laser pulses to selectively produce A? mediated pain and of longer pulses to selectively produce C fiber mediated thermal pain. Thus, the use of these or similar protocols may be useful in developing and testing novel therapeutics based on the differential molecular mechanisms underlying activation of the two fiber types (e.g., TRPV1, TRPV2, etc). In addition, these protocol may be useful in determining the fiber mediation of different clinical pain types which may, in turn be useful in treatment choice.

Abstract

Ablation of TRPV1-expressing nociceptive fibers with the potent capsaicin analog resiniferatoxin (RTX) results in long lasting pain relief. RTX is particularly adaptable to focal application, and the induced chemical axonopathy leads to analgesia with a duration that is influenced by dose, route of administration, and the rate of fiber regeneration. TRPV1 is expressed in a subpopulation of unmyelinated C- and lightly myelinated Adelta fibers that detect changes in skin temperature at low and high rates of noxious heating, respectively. Here we investigate fiber-type specific behaviors, their time course of recovery and molecular correlates of axon damage and nociception using infrared laser stimuli following an RTX-induced peripheral axonopathy.RTX was injected into rat hind paws (mid-plantar) to produce thermal hypoalgesia. An infrared diode laser was used to stimulate Adelta fibers in the paw with a small-diameter (1.6 mm), high-energy, 100 msec pulse, or C-fibers with a wide-diameter (5 mm), long-duration, low-energy pulse. We monitored behavioral responses to indicate loss and regeneration of fibers. At the site of injection, responses to C-fiber stimuli were significantly attenuated for two weeks after 5 or 50 ng RTX. Responses to Adelta stimuli were significantly attenuated for two weeks at the highest intensity stimulus, and for 5 weeks to a less intense Adelta stimulus. Stimulation on the toe, a site distal to the injection, showed significant attenuation of Adelta responses for 7- 8 weeks after 5 ng, or 9-10 weeks after 50 ng RTX. In contrast, responses to C-fiber stimuli exhibited basically normal responses at 5 weeks after RTX. During the period of fiber loss and recovery, molecular markers for nerve regeneration (ATF3 and galanin) are upregulated in the dorsal root ganglia (DRG) when behavior is maximally attenuated, but markers of nociceptive activity (c-Fos in spinal cord and MCP-1 in DRG), although induced immediately after RTX treatment, returned to normal.Behavioral recovery following peripheral RTX treatment is linked to regeneration of TRPV1-expressing Adelta and C-fibers and sustained expression of molecular markers. Infrared laser stimulation is a potentially valuable tool for evaluating the behavioral role of Adelta fibers in pain and pain control.

Abstract

Although nociceptive afferents innervating the body have been heavily studied form many years, much less attention has been paid to trigeminal afferent biology. In particular, very little is known concerning trigeminal nociceptor responses to heat, and almost nothing in the rat. This study uses a highly controlled and reproducible diode laser stimulator to investigate the activation of trigeminal afferents to noxious skin heating.The results of this experiment demonstrate that trigeminal thermonociceptors are distinct from themonociceptors innervating the limbs. Trigeminal nociceptors have considerably slower action potential conduction velocities and lower temperature thresholds than somatic afferent neurons. On the other hand, nociceptors innervating both tissue areas separate into those that respond to short pulse, high rate skin heating and those that respond to long pulse, low rate skin heating.This paper provides the first description in the literature of the in vivo properties of thermonociceptors in rats. These finding of two separate populations aligns with the separation between C and A-delta thermonociceptors innervating the paw, but have significant differences in terms of temperature threshold and average conduction velocities. An understanding of the temperature response properties of afferent neurons innervating the paw skin have been critical in many mechanistic discoveries, some leading to new pain therapies. A clear understanding of trigeminal nociceptors may be similarly useful in the investigation of trigeminal pain mechanisms and potential therapies.

Abstract

An important question remains as to how the brain differentially processes first (pricking) pain mediated by Adelta-nociceptors versus second (burning) pain mediated by C-nociceptors. In the present cross-over randomized, within-subjects controlled study, brain activity patterns were examined with event-related fMRI while pricking and burning pain were selectively evoked using a diode laser. Stimuli evoking equivalent pain intensities were delivered to the dorsum of the left foot. Different laser parameters were used to elicit pricking (60ms pulse duration) and burning (2.0s pulse duration) pain. Whole brain group analysis showed that several brain areas were commonly activated by pricking and burning pain, including bilateral thalamus, bilateral anterior insula, bilateral posterior parietal lobule, contralateral dorsolateral prefrontal cortex, ipsilateral cerebellum, and mid anterior cingulate cortex. These findings show that pricking and burning pain were associated with activity in many of the same nociceptive processing brain regions. This may be expected given that Adelta-and C-nociceptive signals converge to a great extent at the level of the dorsal horn. Other brain regions showed differential processing. Stronger activation in the pricking pain condition was found in the ipsilateral hippocampus, bilateral parahippocampal gyrus, bilateral fusiform gyrus, contralateral cerebellum and contralateral cuneus/parieto-occipital sulcus. Stronger activation in the burning pain condition was found in the ipsilateral dorsolateral prefrontal cortex. These differential activation patterns suggest preferential importance of Adelta-fiber signals versus C-fiber signals for these specific brain regions.

Abstract

Radiant heat is often used for studying thermal nociception, although inherent characteristics such as the broad spectrum of applied wavelengths of typical light sources limit control over and repeatability of stimuli. To overcome these problems, we used a diode infrared laser-based stimulator (wavelength: 980 nm) for selectively stimulating trigeminal Adelta or C thermonociceptors in rats. To provide indirect evidence for nociceptor-selective stimulation, we tested the effects of capsaicin, dimethylsulfoxide (DMSO), and morphine on withdrawal latencies for long pulses with a low current (hypothesized to selectively stimulate C nociceptors) and for threshold currents of short pulses with high current (hypothesized to selectively stimulate Adelta nociceptors) in lightly anesthetized rats. Nonmem analysis was used to perform pharmacodynamic modeling. The measured baseline withdrawal latency for long pulses was 12.5 +/- 0.3 s which was changed significantly to 6.7 +/- 0.4 s after applying topical capsaicin which selectively sensitizes C nociceptors and to 16.5 +/- 1.3 s after 1.0 mg/kg morphine which preferentially attenuates C fiber nociception. Topical DMSO which appears to selectively sensitize Adelta afferents did not significantly alter withdrawal latencies to the long pulses. Fitted threshold currents for short pulses after DMSO were however significantly lower (974 +/- 53 mA vs. 1113 +/- 12 mA for baseline) indicating Adelta sensitization. Capsaicin and morphine did not significantly change threshold currents. Best Nonmem fits for the long pulse were obtained using a model assuming no DMSO effect, but a different inter-individual variability after applying this substance. For the short pulse, a model assuming no capsaicin or morphine effect, but again allowing different inter-individual variabilities after applying these drugs, best described the data. We conclude that different settings of the stimulator used in this study were capable of selectively activating trigeminal Adelta or C thermonociceptors.

Abstract

Radiant heat is often used to study nociception in vivo. We now used infrared radiation generated by a diode laser stimulator (wavelength 980 nm) to investigate transduction mechanisms for noxious heat stimuli in acutely dissociated dorsal root ganglion (DRG) neurons of rats in vitro. The laser stimulator offered the unique opportunity to test whether the same stimuli also elicit pain sensations in humans. A specific heat-induced current (I(heat)) was elicited in six of 13 small DRG neurons (diameter < or =30 microm) tested in the whole-cell configuration of the patch-clamp mode. Current responses in the seven heat-insensitive neurons were within the range explainable by the temperature dependence of the recording setup. I(heat) was characterized by: (1) non-linearity of its amplitude during a suprathreshold heat ramp as well as with stimuli of increasing intensity with an estimated threshold of 42 +/- 1 degrees C; (2) fast rise time and even faster decay time (t(1/2) = 96.5 +/- 5.9 and 27.7 +/- 1.5 ms, respectively); and (3) rate dependence of its induction. All three heat-sensitive neurons tested were also sensitive to capsaicin. The mean threshold for the induction of I(heat) was 2.8 +/- 0.3 J mm(-2). The threshold for the induction of action potentials by depolarizing current pulses was significantly reduced after laser stimulation, suggesting a sensitization at the transformation stage. No such change was seen in heat-insensitive neurons that underwent the same heat stimuli. The same diode laser elicited pain sensations and laser-evoked potentials in human subjects. No significant differences were seen between the pain thresholds in hairy and in glabrous skin, probably due to the deep penetration of this laser radiation. The mean pain threshold for stimuli > or =200 ms in humans was 2.5 +/- 0.2 J mm(-2) (n = 11), and did not differ from the thresholds for the induction of I(heat) in vitro. Our results indicate that I(heat) in primary sensory neurons can be activated by infrared laser pulses that are painful in humans.